Exploitation of synergistic effects between semiochemicals and Metarhizium brunneum against soil pests Dissertation zur Erlangung des Doktorgrades der Fakultät für Agrarwissenschaften der Georg-August-Universität Göttingen vorgelegt von Michael Albert Brandl geboren in Kempten (Allgäu) Göttingen, Mai 2016 _________________________________________________________________ __________ D7 1. Referent: Prof. Dr. Stefan Vidal 2. Referent: Prof. Dr. Anant Patel Tag der mündlichen Prüfung: 13. Mai 2015 Ich danke dir, Herr, mein Gott, von ganzem Herzen und ehre deinen Namen ewiglich. Psalm 86,12 Table of contents Summary ................................................................................................................ 1 General introduction ............................................................................................... 3 Objectives............................................................................................................... 8 Chapter 1 ............................................................................................................... 9 Screening of botanical extracts for repellence against western corn rootworm larvae ..................................................................................................... Chapter 2 ............................................................................................................. 10 A stress-and-kill approach using a combination of turmeric and Metarhizium brunneum for western corn rootworm larval control .......................................... Abstract ................................................................................................................ 11 Introduction........................................................................................................... 12 Materials and methods ......................................................................................... 13 Results ................................................................................................................. 21 Discussion ............................................................................................................ 26 Conclusion............................................................................................................ 29 Acknowledgements .............................................................................................. 29 References ........................................................................................................... 30 Chapter 3 ............................................................................................................. 37 Wireworm damage reduction in potatoes with an attract-and-kill strategy using Metarhizium brunneum ............................................................................... Abstract ................................................................................................................ 38 Introduction........................................................................................................... 39 Materials and methods ......................................................................................... 41 Results ................................................................................................................. 48 Discussion ............................................................................................................ 57 Conclusion............................................................................................................ 62 Acknowledgements .............................................................................................. 63 References ........................................................................................................... 63 Chapter 4 ............................................................................................................. 73 Technical attract-and-kill formulations for wireworm control with Metarhizium brunneum in potatoes ...................................................................... Abstract ................................................................................................................ 74 Introduction........................................................................................................... 75 Materials and methods ......................................................................................... 77 Results ................................................................................................................. 84 Discussion ............................................................................................................ 92 Conclusion............................................................................................................ 96 Acknowledgements .............................................................................................. 96 References ........................................................................................................... 97 General discussion ............................................................................................. 105 Conclusion.......................................................................................................... 110 References: General introduction and general discussion ................................. 111 Publications ........................................................................................................ 119 Acknowledgements / Danksagung ..................................................................... 121 Summary Summary Root-feeding insects, such as the larvae of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) and wireworms Agriotes spp. Eschscholtz (Coleoptera: Elateridae), the larvae of click beetles, are widely recognized as major soil pests, which influence agricultural productivity. Environmental concerns and a high risk of insecticide use as well as the lack of other efficient control options require new and effective strategies to target these pests. Two behavioral based control approaches, combining the use of the entomopathogenic fungus Metarhizium brunneum (Metschnikoff) Sorokin from the order Hypocreales (Ascomycota) and semiochemicals as efficacy enhancing agents, were tested to improve biological soil pest control. WCR larval control was evaluated with a “stress-and-kill” (SK) strategy in laboratory and greenhouse experiments using botanical repellents to increase the chance of contact and susceptibility with M. brunneum conidia. Wireworm control was tested with an “attract-and-kill” (AK) strategy in field and laboratory experiments, whereas the pest is lured towards M. brunneum conidia with an artificial carbon dioxide (CO2) source as an attractant. Both strategies were conducted with different formulation techniques to improve shelf-life and practical feasibility for an application in the field. 1. The behavioral and spatial distribution changes of second instar WCR larvae were studied in no-choice tests conducted in Petri dish bioassays and rhizotron experiments to assess the most suitable botanical extract as a stressing agent. a. Garlic and turmeric derived extracts were evaluated as the most promising extracts as WCR larvae avoided garlic / turmeric treated soil. b. Turmeric evaluation in rhizotron experiments revealed WCR larval avoidance of turmeric soil up to 5 cm from its application point in the maize (Zea mays L.) root system resulting in an increased dispersal and larval movement in the soil. c. Turmeric was therefore evaluated as a suitable “stressing” agent for WCR larvae 1 Summary 2. The performance of a SK strategy against neonate WCR larvae was studied in maize planted small containers in the greenhouse to assess potential synergies between formulated M. brunneum conidia and turmeric. a. Repellent effects of turmeric were demonstrated against neonate WCR larvae b. Turmeric did not impact M. brunneum establishment and growth, excluding any negative side effects through turmeric’s antifungal properties with a combination of both SK agents. c. The application of wet M. brunneum formulated beads only or in combination with turmeric failed in larval control. SK is therefore not feasible for WCR control due to the lack of WCR larval control with M. brunneum alone. 3. The performance of an AK strategy against wireworms (Agriotes spp.) was studied under field conditions in organic potato fields across three field seasons. The first two field seasons looked into the interaction between CO2 and M. brunneum to evaluate the enhancement of wireworm control with CO2. Each AK agent was encapsulated in a wet bead and then tested as a co-application through the mixture of “attract” and “kill” beads. The third field season tested a wet and technical dry co-formulation with both AK agents encapsulated within one bead to examine a more practical AK formulation for farmers. The performance of AK agents was tested in different field soil types to assess CO2 emission and M. brunneum virulence and growth. a. CO2 levels in soil increased significantly over 4 – 6 weeks with an application of AK beads. M. brunneum virulence and growth was measured in all tested soil types, but was dependent on the type of AK bead used. b. The combination of CO2 and M. brunneum enhanced wireworm control by up to 35% compared to a M. brunneum treatment alone, but only as a spot application underneath the seed potatoes. A band application of beads could